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Increasing endogenous retinoic acid inhibits leiomyoma cell proliferation
shaped morphology. The growth of the leiomyomas was observed to be more dyregulated as compared to myometrial cells, though no significant difference was observed in a-actin fluorescence intensity. On the synthesized Algimatrix, at the end of 3 weeks of growth, small rounded bodies were observed in leiomyoma plates. The cells were viable as confirmed by Almar blue dye method. CONCLUSIONS: We developed multiple 3-dimensional cultures of human leiomyoma and patient matched myometrial cells. This advance will allow novel investigations on ECM formation and regulation, the component that makes up the bulk of leiomyomas and is central to symptoms. Supported by: Intramural grant, USUHS.
P-138 Tuesday, October 20, 2009 INCREASING ENDOGENOUS RETINOIC ACID INHIBITS LEIOMYOMA CELL PROLIFERATION. W. H. Catherino, T. Delgado, M. Malik, J. Britten, J. Segars, D. McCarthy-Keith. Obstetrics and Gynecology, Uniformed Services of the Health Sciences, Bethesda, MD; Program in Reproductive and Adult Endocrinology, NICHD, NIH, Bethesda, MD. OBJECTIVE: In tissue and leiomyoma cells, we previously identified molecular alterations in the retinoic acid (RA) pathway including increased expression of cytochrome P450 26A1 (CYP26A1) which results in decreased intracellular all-trans retinoic acid (ATRA). Exogenous ATRA inhibited proliferation and extracellular matrix production, but systemic use of ATRA therapeutically for women with leiomyomas could be associated with significant toxicity. Liarozole inhibits CYP26A1 activity, resulting in increased intracellular ATRA. We hypothesized that liarozole treatment would inhibit proliferation of leiomyoma cells in a manner comparable to ATRA. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: Proliferation studies were performed using immortalized human leiomyoma and patient-matched myometrial cells treated with liarozole, ATRA or combination of both at concentrations ranging from 10uM to 100uM. Proliferation was measured using the Sulforhodamine B method. Statistical evaluation was performed using the Wilcoxon signed-rank test, with significance set at p<0.05. RESULTS: All-trans retinoic acid inhibited leiomyoma cell proliferation at 2.5uM and had further inhibition in a dose-dependent manner. Liarozole treatment resulted in a dose-dependent decrease of leiomyoma cell proliferation to 57% of control 2% at 100uM. Statistically significant decreases were identified at liarozole concentrations of 25uM and higher. Combination studies were also performed using ATRA at 5uM and 10uM and a range of liarozole concentrations from 10uM to 100uM. There was a dose-dependent decrease of leiomyoma cell proliferation to 20% of control 1% at 100uM of liarozole and 10uM of ATRA, demonstrating a synergistic effect. Statistically significant decreases were identified at concentrations as low as 10uM of liarozole and 5uM of ATRA. CONCLUSIONS: Liarozole inhibits leiomyoma cell proliferation, and acts synergistically with exogenous ATRA. Liarozole represents a novel class of therapies for the treatment of uterine leiomyomas. Supported by: The AAOGF.
P-139 Tuesday, October 20, 2009 PROLIFERATION OF UTERINE FIBROIDS IN HYPOXIA IS ASSOCIATED WITH INCREASED ENDOTHELIN-1. K. Wallace, E. Turnage, L. F. Ray, B. D. Cowan, B. LaMarca. Obstetrics and Gynecology, University of Mississippi Medical Center, Jackson, MS. OBJECTIVE: Uterine leiomyomas substantially reduce quality-of-life in affected women. Independent observations suggest an association of a hypoxic drive that initiates fibroid development. A role for the vasoactive peptide, Endothelin-1 (ET-1), was recently demonstrated by other laboratories to inhibit apoptosis of leiomyoma cells in vitro. This study was designed to address the hypothesis that uterine fibroid proliferation is associated with production of the vasoactive peptide ET-1, and ET-1 production may serve as a link between uterine fibroid proliferation and the development of hypertension in women. DESIGN: The design of this study used an in vitro hypoxic model of fibroid and myometrium tissue culture to determine if uterine fibroids are a source of ET-1, and if hypoxia exacerbates ET-1 secretion from mesenchymal tissues. MATERIALS AND METHODS: Immediately following hysterectomy, fibroids and myometrium were excised from uterus (IRB approved), washed in
FERTILITY & STERILITYÒ
cold PBS, and approximately 200-400 U thickness tissue slices were plated on matrigel-coated well inserts with media (DMEM, 10% FCS). To induce hypoxia, cultures were placed in a modular incubator chamber with two final pO2 concentrations of 1 and 6%. ET-1, sFLT (vasoactive), and IL-6 (control) were measured from cell culture supernatants. RESULTS: Six and seven tissue samples were tested against 1% and 6% oxygen, respectively. A fibroid/myometrium ratio of the tested molecules generated an increase of ET-1 at 1% oxygen (ratio of fibroid/myometrium¼1.310.12 {SEM}, [95% CI;1.07,1.68]). All other tested molecules had overlapping 95% CI, and t-test of each pair of molecules at 1% and 6% oxygen showed no difference. CONCLUSIONS: Uterine fibroids are a source of ET-1 production, and extreme hypoxia stimulated changes in EF-1 from human uterine lieomyomas. These data suggest a possible link between vasoactive peptides and development or growth of uterine fibroids, and offers a potential molecular pathway for the treatment of uterine fibroids. Supported by: Supported by the ObGyn Foundation.
P-140 Tuesday, October 20, 2009 A PROSPECTIVE CASE-MATCHED COHORT ANALYSIS OF MAGNETIC RESONANCE-GUIDED FOCUSED ULTRASOUND VERSUS UTERINE ARTERY EMBOLIZATION FOR TREATMENT OF SYMPTOMATIC UTERINE FIBROIDS. L. N. C. Johnson, M. L. Leong, M. J. Miller, M. A. Behera. Obstetrics and Gynecology, Duke University Medical Center, Durham, NC; Radiology, Duke University Medical Center, Durham, NC. OBJECTIVE: To compare quality of life outcomes in patients who underwent magnetic resonance-guided focused ultrasound (MRgFUS) or uterine artery embolization (UAE) for treatment of symptomatic uterine fibroids. DESIGN: A prospective case-matched cohort analysis was performed. Fifteen patients who underwent MRgFUS were compared with 30 patients who underwent UAE from January 2007 to March 2009. Patients were first matched by age and then by size of largest fibroid. The primary endpoint was patients’ scores pre- and post-procedure on the Uterine Fibroid Symptom and Health-Related Quality of Life Questionnaire (UFS-QOL). Secondary endpoints were the need for further treatment and patient satisfaction with treatment. MATERIALS AND METHODS: Patient characteristics and pre-procedure quality of life scores were obtained by chart review. Patients were contacted by telephone for post-procedure quality of life scores and missing data. Means and 95% confidence intervals were calculated. RESULTS: Of patients who completed the UFS-QOL before and after treatment, 100% (10/10) in the MRgFUS group reported a R10 point decrease in the Symptom Severity Score (SSS) compared to 87% (20/23) of patients in the UAE group. After treatment, UAE patients scored significantly higher than MRgFUS patients on four out of the eight UFS-QOL subscores: Concern, Self-consciousness, Sexual function, and Total health related quality of life. Patients in the MRgFUS group had significantly larger fibroids (8.6 2.0 cm) than UAE patients (6.8 2.5 cm, p¼0.0131). CONCLUSIONS: Both MRgFUS and UAE are effective, uterine-sparing fibroid treatment options that demonstrate significant symptomatic improvement in quality of life scores as defined by a 10 point decrease in SSS on the UFS-QOL. Further studies are needed to determine if clinical outcomes are impacted by patient selection and fibroid characteristics. In this study, women who chose to have MRgFUS had significantly larger fibroids, which may have contributed to a more modest improvement in symptom scores.
P-141 Tuesday, October 20, 2009 1, 25 DIHYDROXYVITAMIN D3 DISORGANIZES ACTIN FIBERS IN HUMAN IMMORTALIZED UTERINE LEIOMYOMA CELLS. S. K. Halder, C. Sharan, S. Goodwin, A. Al-Hendy. Obstetrics and Gynecology, Meharry Medical College, Nashville, TN; Cancer Biology, Meharry Medical College, Nashville, TN. OBJECTIVE: Leiomyomas (uterine fibroids) are benign tumors in uterus of reproductive age women with increasing synthesis and accumulation of fibrotic gene products and are often associated with infertility. We will evaluate whether biologically active 1, 25 dihydroxyvitamin D3 (Vitamin D) affect fibrotic gene expressions in immortalized human uterine leiomyoma (HuLM) cells. DESIGN: We use in vitro HuLM cell culture to evaluate fibrosis.
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P-138 Tuesday, October 20, 2009 INCREASING ENDOGENOUS RETINOIC ACID INHIBITS LEIOMYOMA CELL PROLIFERATION. W. H. Catherino, T. Delgado, M. Malik, J. Britten, J. Segars, D. McCarthy-Keith. Obstetrics and Gynecology, Uniformed Services of the Health Sciences, Bethesda, MD; Program in Reproductive and Adult Endocrinology, NICHD, NIH, Bethesda, MD. OBJECTIVE: In tissue and leiomyoma cells, we previously identified molecular alterations in the retinoic acid (RA) pathway including increased expression of cytochrome P450 26A1 (CYP26A1) which results in decreased intracellular all-trans retinoic acid (ATRA). Exogenous ATRA inhibited proliferation and extracellular matrix production, but systemic use of ATRA therapeutically for women with leiomyomas could be associated with significant toxicity. Liarozole inhibits CYP26A1 activity, resulting in increased intracellular ATRA. We hypothesized that liarozole treatment would inhibit proliferation of leiomyoma cells in a manner comparable to ATRA. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: Proliferation studies were performed using immortalized human leiomyoma and patient-matched myometrial cells treated with liarozole, ATRA or combination of both at concentrations ranging from 10uM to 100uM. Proliferation was measured using the Sulforhodamine B method. Statistical evaluation was performed using the Wilcoxon signed-rank test, with significance set at p<0.05. RESULTS: All-trans retinoic acid inhibited leiomyoma cell proliferation at 2.5uM and had further inhibition in a dose-dependent manner. Liarozole treatment resulted in a dose-dependent decrease of leiomyoma cell proliferation to 57% of control 2% at 100uM. Statistically significant decreases were identified at liarozole concentrations of 25uM and higher. Combination studies were also performed using ATRA at 5uM and 10uM and a range of liarozole concentrations from 10uM to 100uM. There was a dose-dependent decrease of leiomyoma cell proliferation to 20% of control 1% at 100uM of liarozole and 10uM of ATRA, demonstrating a synergistic effect. Statistically significant decreases were identified at concentrations as low as 10uM of liarozole and 5uM of ATRA. CONCLUSIONS: Liarozole inhibits leiomyoma cell proliferation, and acts synergistically with exogenous ATRA. Liarozole represents a novel class of therapies for the treatment of uterine leiomyomas. Supported by: The AAOGF.
P-139 Tuesday, October 20, 2009 PROLIFERATION OF UTERINE FIBROIDS IN HYPOXIA IS ASSOCIATED WITH INCREASED ENDOTHELIN-1. K. Wallace, E. Turnage, L. F. Ray, B. D. Cowan, B. LaMarca. Obstetrics and Gynecology, University of Mississippi Medical Center, Jackson, MS. OBJECTIVE: Uterine leiomyomas substantially reduce quality-of-life in affected women. Independent observations suggest an association of a hypoxic drive that initiates fibroid development. A role for the vasoactive peptide, Endothelin-1 (ET-1), was recently demonstrated by other laboratories to inhibit apoptosis of leiomyoma cells in vitro. This study was designed to address the hypothesis that uterine fibroid proliferation is associated with production of the vasoactive peptide ET-1, and ET-1 production may serve as a link between uterine fibroid proliferation and the development of hypertension in women. DESIGN: The design of this study used an in vitro hypoxic model of fibroid and myometrium tissue culture to determine if uterine fibroids are a source of ET-1, and if hypoxia exacerbates ET-1 secretion from mesenchymal tissues. MATERIALS AND METHODS: Immediately following hysterectomy, fibroids and myometrium were excised from uterus (IRB approved), washed in
FERTILITY & STERILITYÒ
cold PBS, and approximately 200-400 U thickness tissue slices were plated on matrigel-coated well inserts with media (DMEM, 10% FCS). To induce hypoxia, cultures were placed in a modular incubator chamber with two final pO2 concentrations of 1 and 6%. ET-1, sFLT (vasoactive), and IL-6 (control) were measured from cell culture supernatants. RESULTS: Six and seven tissue samples were tested against 1% and 6% oxygen, respectively. A fibroid/myometrium ratio of the tested molecules generated an increase of ET-1 at 1% oxygen (ratio of fibroid/myometrium¼1.310.12 {SEM}, [95% CI;1.07,1.68]). All other tested molecules had overlapping 95% CI, and t-test of each pair of molecules at 1% and 6% oxygen showed no difference. CONCLUSIONS: Uterine fibroids are a source of ET-1 production, and extreme hypoxia stimulated changes in EF-1 from human uterine lieomyomas. These data suggest a possible link between vasoactive peptides and development or growth of uterine fibroids, and offers a potential molecular pathway for the treatment of uterine fibroids. Supported by: Supported by the ObGyn Foundation.
P-140 Tuesday, October 20, 2009 A PROSPECTIVE CASE-MATCHED COHORT ANALYSIS OF MAGNETIC RESONANCE-GUIDED FOCUSED ULTRASOUND VERSUS UTERINE ARTERY EMBOLIZATION FOR TREATMENT OF SYMPTOMATIC UTERINE FIBROIDS. L. N. C. Johnson, M. L. Leong, M. J. Miller, M. A. Behera. Obstetrics and Gynecology, Duke University Medical Center, Durham, NC; Radiology, Duke University Medical Center, Durham, NC. OBJECTIVE: To compare quality of life outcomes in patients who underwent magnetic resonance-guided focused ultrasound (MRgFUS) or uterine artery embolization (UAE) for treatment of symptomatic uterine fibroids. DESIGN: A prospective case-matched cohort analysis was performed. Fifteen patients who underwent MRgFUS were compared with 30 patients who underwent UAE from January 2007 to March 2009. Patients were first matched by age and then by size of largest fibroid. The primary endpoint was patients’ scores pre- and post-procedure on the Uterine Fibroid Symptom and Health-Related Quality of Life Questionnaire (UFS-QOL). Secondary endpoints were the need for further treatment and patient satisfaction with treatment. MATERIALS AND METHODS: Patient characteristics and pre-procedure quality of life scores were obtained by chart review. Patients were contacted by telephone for post-procedure quality of life scores and missing data. Means and 95% confidence intervals were calculated. RESULTS: Of patients who completed the UFS-QOL before and after treatment, 100% (10/10) in the MRgFUS group reported a R10 point decrease in the Symptom Severity Score (SSS) compared to 87% (20/23) of patients in the UAE group. After treatment, UAE patients scored significantly higher than MRgFUS patients on four out of the eight UFS-QOL subscores: Concern, Self-consciousness, Sexual function, and Total health related quality of life. Patients in the MRgFUS group had significantly larger fibroids (8.6 2.0 cm) than UAE patients (6.8 2.5 cm, p¼0.0131). CONCLUSIONS: Both MRgFUS and UAE are effective, uterine-sparing fibroid treatment options that demonstrate significant symptomatic improvement in quality of life scores as defined by a 10 point decrease in SSS on the UFS-QOL. Further studies are needed to determine if clinical outcomes are impacted by patient selection and fibroid characteristics. In this study, women who chose to have MRgFUS had significantly larger fibroids, which may have contributed to a more modest improvement in symptom scores.
P-141 Tuesday, October 20, 2009 1, 25 DIHYDROXYVITAMIN D3 DISORGANIZES ACTIN FIBERS IN HUMAN IMMORTALIZED UTERINE LEIOMYOMA CELLS. S. K. Halder, C. Sharan, S. Goodwin, A. Al-Hendy. Obstetrics and Gynecology, Meharry Medical College, Nashville, TN; Cancer Biology, Meharry Medical College, Nashville, TN. OBJECTIVE: Leiomyomas (uterine fibroids) are benign tumors in uterus of reproductive age women with increasing synthesis and accumulation of fibrotic gene products and are often associated with infertility. We will evaluate whether biologically active 1, 25 dihydroxyvitamin D3 (Vitamin D) affect fibrotic gene expressions in immortalized human uterine leiomyoma (HuLM) cells. DESIGN: We use in vitro HuLM cell culture to evaluate fibrosis.
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